Code used to generate paper results : Database comparison and CT characteristics
Julie Devis
2023-08-28
1 Loading data and packages
library(readxl)
library(readr)
library(CTexploreR)
library(Vennerable)
library(biomaRt)
library(tidyverse)
library(SummarizedExperiment)
library(UpSetR)
library(ComplexHeatmap)
library(circlize)
library(SingleCellExperiment)
library(org.Hs.eg.db)
library(clusterProfiler)
library(msigdbr)
library(DOSE)
library(BiocParallel)
library(patchwork)
library(Biostrings)Gene names/synonyms required for databases cleaning
ensembl <- biomaRt::useDataset("hsapiens_gene_ensembl", useMart("ensembl"))
attributes_vector <- c("ensembl_gene_id", "external_gene_name",
"external_synonym", "gene_biotype",
"chromosome_name", "band", "start_position", "end_position",
"strand")
ensembl_gene_synonym <- as_tibble(getBM(attributes = attributes_vector, mart = ensembl))
ensembl_gene_synonym <- ensembl_gene_synonym %>%
mutate(external_synonym = sub(pattern = "ORF", external_synonym,
replacement = "orf"))
attributes_vector <- c("ensembl_gene_id", "external_gene_name")
ensembl_gene_names <- as_tibble(getBM(attributes = attributes_vector, mart = ensembl))GTEX_data <- CTdata::GTEX_data()## Error in readRDS(file) : error reading from connectionnormal_tissues_multimapping_data <- CTdata::normal_tissues_multimapping_data()
CCLE_data <- CTdata::CCLE_data()
TCGA_TPM <- CTdata::TCGA_TPM()
DAC_treated_cells_multimapping <- CTdata::DAC_treated_cells_multimapping()
testis_sce <- CTdata::testis_sce()
scRNAseq_HPA <- CTdata::scRNAseq_HPA()
CT_mean_methylation_in_tissues <- CTdata::CT_mean_methylation_in_tissues()Common figures parameters
legends_param <- list(
labels_gp = gpar(col = "black", fontsize = 6),
title_gp = gpar(col = "black", fontsize = 6),
row_names_gp = gpar(fontsize = 4),
annotation_name_side = "left")
legend_colors <- c("#5E4FA2", "#3288BD", "#66C2A5", "#ABDDA4", "#E6F598",
"#FFFFBF", "#FEE08B", "#FDAE61", "#F46D43", "#D53E4F",
"#9E0142")
chr_colors <- c("X-linked" = "deeppink", "Not X" = "royalblue1")
meth_colors <- c("Methylation" = "lightgreen", "Not methylation" = "gray")2 Database comparison
2.1 Lists cleaning
CT lists from other databases have been checked (using GTEx and our
GTEx_expression() funtion and GeneCards) in order to remove
duplicated gene names or deprecated ones and allow comparison between
databases.
2.1.1 CTdatabase
Online list copied in a csv file, several lists exist so we combined them.
We checked gene names that were a concetanation of two genes (choice using biomaRt synonyms to get the official one), checked which ones had the right names, removed duplicated genes, verified lost genes and added back those that should be there.
CTdatabase <- read_delim("data/CTdatabase1.csv", delim = ";",
escape_double = FALSE, trim_ws = TRUE)
colnames(CTdatabase) <- c("Family", "Gene_Name", "Chromosomal_localization",
"CT_identifier")
CTdatabase_bis <- read_csv2("data/CTdatabase2.csv")
CTdatabase <- left_join(CTdatabase, CTdatabase_bis,
by = c("Gene_Name" = "Gene_Symbol"))
CTdatabase_single <- CTdatabase %>%
mutate(Gene_Name = sub(pattern = "/.*$", Gene_Name, replacement = ""))
CTdatabase_single <- CTdatabase_single %>%
mutate(Gene_Name = sub(pattern = ",.*$", Gene_Name, replacement = ""))
CTdatabase_official_names <-
unique(dplyr::select(ensembl_gene_synonym, ensembl_gene_id,
external_gene_name)) %>%
filter(external_gene_name %in% CTdatabase_single$Gene_Name) %>%
mutate(Gene_Name = external_gene_name) %>%
mutate(external_synonym = NA)
CTdatabase_synonym <-
ensembl_gene_synonym %>%
filter(external_synonym %in% CTdatabase_single$Gene_Name) %>%
mutate(Gene_Name = external_synonym) %>%
dplyr::select(ensembl_gene_id, external_gene_name, Gene_Name, external_synonym)
CTdatabase_cleaned <-
rbind(CTdatabase_official_names, CTdatabase_synonym) %>%
left_join(CTdatabase_single)
duplicated_genes <- CTdatabase_cleaned$Gene_Name[duplicated(CTdatabase_cleaned$Gene_Name)]
bad_ids <- ensembl_gene_synonym %>%
filter(external_gene_name %in% duplicated_genes | external_synonym %in% duplicated_genes) %>%
filter(chromosome_name %in% grep(pattern = "H", x = chromosome_name, value = TRUE)) %>%
pull(ensembl_gene_id)
CTdatabase_cleaned <- CTdatabase_cleaned %>%
dplyr::filter(!ensembl_gene_id %in% bad_ids)
CTdatabase_cleaned <- CTdatabase_cleaned %>%
filter(!ensembl_gene_id == "ENSG00000052126")
CTdatabase_cleaned <- CTdatabase_cleaned %>%
filter(!(ensembl_gene_id == "ENSG00000183305" & Gene_Name == "MAGEA2"))
CTdatabase_cleaned <- CTdatabase_cleaned %>%
filter(!ensembl_gene_id == "ENSG00000204648")
CTdatabase_cleaned <- filter(CTdatabase_cleaned, Gene_Name != "CSAG3B")
CTdatabase_cleaned[CTdatabase_cleaned$Gene_Name == "CSAG2", "external_synonym"] <- "CSAG3B"
CTdatabase_cleaned <- filter(CTdatabase_cleaned, Gene_Name != "CT45A4")
CTdatabase_cleaned[CTdatabase_cleaned$Gene_Name == "CT45A3", "external_synonym"] <- "CT45A4"
CTdatabase_cleaned <- filter(CTdatabase_cleaned, Gene_Name != "LAGE-1b")
CTdatabase_cleaned[CTdatabase_cleaned$Gene_Name == "CTAG2", "external_synonym"] <- "LAGE-1b"
CTdatabase_cleaned <- filter(CTdatabase_cleaned, Gene_Name != "CT16.2")
CTdatabase_cleaned[CTdatabase_cleaned$Gene_Name == "PAGE5", "external_synonym"] <- "CT16.2"
CTdatabase_cleaned <- filter(CTdatabase_cleaned, Gene_Name != "SPANXB2")
CTdatabase_cleaned[CTdatabase_cleaned$Gene_Name == "SPANXB1", "external_synonym"] <- "SPANXB2"
CTdatabase_cleaned <- filter(CTdatabase_cleaned, Gene_Name != "SPANXE")
CTdatabase_cleaned[CTdatabase_cleaned$Gene_Name == "SPANXD", "external_synonym"] <- "SPANXE"
CTdatabase_cleaned <- filter(CTdatabase_cleaned, Gene_Name != "XAGE1C")
CTdatabase_cleaned <- filter(CTdatabase_cleaned, Gene_Name != "XAGE1D")
CTdatabase_cleaned <- filter(CTdatabase_cleaned, Gene_Name != "XAGE1E")
CTdatabase_cleaned <- filter(CTdatabase_cleaned, Gene_Name != "XAGE2B")
CTdatabase_cleaned[CTdatabase_cleaned$Gene_Name == "XAGE2", "external_synonym"] <- "XAGE2B"
CTdatabase_cleaned <- filter(CTdatabase_cleaned, Gene_Name != "CTAGE-2")
CTdatabase_cleaned[CTdatabase_cleaned$Gene_Name == "CTAGE1", "external_synonym"] <- "CTAGE-2"
CTdatabase_cleaned <- ensembl_gene_synonym %>%
mutate(Gene_Name = external_synonym) %>%
filter(external_synonym == "CXorf61") %>%
dplyr::select(ensembl_gene_id, external_gene_name, Gene_Name, external_synonym) %>%
cbind(CTdatabase_single[CTdatabase_single$Gene_Name == "Cxorf61",
c("Family", "Chromosomal_localization", "CT_identifier", "Classification")]) %>%
rbind(CTdatabase_cleaned)
CTdatabase_cleaned <- unique(dplyr::select(ensembl_gene_synonym, ensembl_gene_id, external_gene_name)) %>%
filter(external_gene_name == "CCNA1") %>%
mutate(Gene_Name = external_gene_name) %>%
mutate(external_synonym = NA) %>%
cbind(CTdatabase_single[CTdatabase_single$Gene_Name == "cyclin A1",
c("Family", "Chromosomal_localization", "CT_identifier", "Classification")]) %>%
rbind(CTdatabase_cleaned)
CTdatabase_cleaned <- unique(dplyr::select(ensembl_gene_synonym, ensembl_gene_id, external_gene_name))%>%
filter(external_gene_name == "GOLGA6L2") %>%
filter(ensembl_gene_id == "ENSG00000174450") %>%
mutate(Gene_Name = external_gene_name) %>%
mutate(external_synonym = NA) %>%
cbind(CTdatabase_single[CTdatabase_single$Gene_Name == "GOLGAGL2 FA",
c("Family", "Chromosomal_localization", "CT_identifier", "Classification")]) %>%
rbind(CTdatabase_cleaned)
CTdatabase_cleaned <- unique(dplyr::select(ensembl_gene_synonym, ensembl_gene_id, external_gene_name))%>%
filter(external_gene_name == "LYPD6B") %>%
mutate(Gene_Name = external_gene_name) %>%
mutate(external_synonym = NA) %>%
cbind(CTdatabase_single[CTdatabase_single$Gene_Name == "LOC130576",
c("Family", "Chromosomal_localization", "CT_identifier", "Classification")]) %>%
rbind(CTdatabase_cleaned)
CTdatabase_cleaned <- unique(dplyr::select(ensembl_gene_synonym, ensembl_gene_id, external_gene_name))%>%
filter(external_gene_name == "CT62") %>%
mutate(Gene_Name = external_gene_name) %>%
mutate(external_synonym = NA) %>%
cbind(CTdatabase_single[CTdatabase_single$Gene_Name == "LOC196993",
c("Family", "Chromosomal_localization", "CT_identifier", "Classification")]) %>%
rbind(CTdatabase_cleaned)
CTdatabase_cleaned <- unique(dplyr::select(ensembl_gene_synonym, ensembl_gene_id, external_gene_name))%>%
filter(external_gene_name == "CT75") %>%
filter(ensembl_gene_id == "ENSG00000291155") %>%
mutate(Gene_Name = external_gene_name) %>%
mutate(external_synonym = NA) %>%
cbind(CTdatabase_single[CTdatabase_single$Gene_Name == "LOC440934",
c("Family", "Chromosomal_localization", "CT_identifier", "Classification")]) %>%
rbind(CTdatabase_cleaned)
CTdatabase_cleaned <- unique(dplyr::select(ensembl_gene_synonym, ensembl_gene_id, external_gene_name))%>%
filter(external_gene_name == "LINC01192") %>%
mutate(Gene_Name = external_gene_name) %>%
mutate(external_synonym = NA) %>%
cbind(CTdatabase_single[CTdatabase_single$Gene_Name == "LOC647107",
c("Family", "Chromosomal_localization", "CT_identifier", "Classification")]) %>%
rbind(CTdatabase_cleaned)
CTdatabase_cleaned <- unique(dplyr::select(ensembl_gene_synonym, ensembl_gene_id, external_gene_name))%>%
filter(external_gene_name == "TSPY1") %>%
mutate(Gene_Name = external_gene_name) %>%
mutate(external_synonym = NA) %>%
cbind(CTdatabase_single[CTdatabase_single$Gene_Name == "LOC728137",
c("Family", "Chromosomal_localization", "CT_identifier", "Classification")]) %>%
rbind(CTdatabase_cleaned)
CTdatabase_cleaned <- unique(dplyr::select(ensembl_gene_synonym, ensembl_gene_id, external_gene_name))%>%
filter(external_gene_name == "SSX2B") %>%
mutate(Gene_Name = external_gene_name) %>%
mutate(external_synonym = NA) %>%
cbind(CTdatabase_single[CTdatabase_single$Gene_Name == "SSX2b",
c("Family", "Chromosomal_localization", "CT_identifier", "Classification")]) %>%
rbind(CTdatabase_cleaned)2.1.2 Jamin’s list
Excel file coming from supplemental data.
Jamin_core_CT <- read_excel("data/Jamin_core_CT.xlsx")
Jamin_core_CT[Jamin_core_CT$Gene == "KIAA1211", "Gene"] <- "CRACD"
Jamin_core_CT[Jamin_core_CT$Gene == "CXorf67", "Gene"] <- "EZHIP"2.1.3 Wang’s CTatlas
Excel file coming from supplemental data.
Wang_CT <- read_excel("data/Wang_Suppl_Data_3.xlsx",
sheet = "Supplementary Data 3B", skip = 1)
colnames(Wang_CT)[1] <- "ensembl_gene_id"
Wang_CT <- ensembl_gene_names %>%
filter(ensembl_gene_id %in% Wang_CT$ensembl_gene_id) %>%
right_join(Wang_CT)
Wang_CT[Wang_CT$ensembl_gene_id == "ENSG00000181013", "external_gene_name"] <- "C17orf47"
Wang_CT[Wang_CT$ensembl_gene_id == "ENSG00000204293", "external_gene_name"] <- "OR8B2"
Wang_CT[Wang_CT$external_gene_name == "", "external_gene_name"] <- "RNASE11"
Wang_CT[Wang_CT$external_gene_name == "CHCT1", "external_gene_name"] <- "C17orf64"
Wang_CT[Wang_CT$external_gene_name == "PRSS40A", "external_gene_name"] <- "TISP43"
Wang_CT[Wang_CT$external_gene_name == "TEX56P", "external_gene_name"] <- "C6orf201"
Wang_CT[Wang_CT$external_gene_name == "SLC25A51P4", "external_gene_name"] <- "RP11-113D6.10"
Wang_CT[Wang_CT$external_gene_name == "TCP10L3", "external_gene_name"] <- "TCP10"
Wang_CT[Wang_CT$external_gene_name == "SCAND3", "external_gene_name"] <- "ZBED9"2.1.4 Carter’s list
Carter_CT_list <- read_table("data/Carter_CT_list.txt", skip = 1)
Carter_CT <- Carter_CT_list[Carter_CT_list$CT_Expression,]
Carter_CT[Carter_CT$Gene == "ENSG00000261649", "Gene_Name"] <- "GOLGA6L7"
Carter_CT[Carter_CT$Gene == "ENSG00000239620", "Gene_Name"] <- "PRR20G"
Carter_CT[Carter_CT$Gene == "ENSG00000168148", "Gene_Name"] <- "H3-4"
Carter_CT[Carter_CT$Gene == "ENSG00000204296", "Gene_Name"] <- "TSBP1"
Carter_CT[Carter_CT$Gene == "ENSG00000180219", "Gene_Name"] <- "GARIN6"
Carter_CT[Carter_CT$Gene == "ENSG00000172717", "Gene_Name"] <- "GARIN2"
Carter_CT[Carter_CT$Gene == "ENSG00000174015", "Gene_Name"] <- "CBY2"
Carter_CT[Carter_CT$Gene == "ENSG00000224960", "Gene_Name"] <- "PPP4R3C"2.2 CTexploreR data for selection pipeline
To characterise the differences between our database and other, we need the category we created in CTexploreR.
Hereunder is what we used for our selection pipeline (coming from
make_CT_list.R in CTdata), mainly how we
created the data.
# GTEX data with the tissue specificity category determined
all_genes <- as_tibble(rowData(GTEX_data), rownames = "ensembl_gene_id")
all_genes$TPM_testis <- assay(GTEX_data)[, "Testis"]
# Add multimapping_analysis assessing testis-specificity of genes "lowly_expressed"
all_genes <- all_genes %>%
left_join(as_tibble(rowData(normal_tissues_multimapping_data),
rownames = "ensembl_gene_id"))
# Summarise both specificity
all_genes <- all_genes %>%
mutate(testis_specificity = case_when(
GTEX_category == "testis_specific" |
multimapping_analysis == "testis_specific" ~ "testis_specific",
GTEX_category == "testis_preferential" ~ "testis_preferential"))
# Add testis scRNA seq highest expressed cell type
all_genes <- all_genes %>%
left_join(as_tibble(rowData(testis_sce)) %>%
dplyr::select(external_gene_name, testis_cell_type))
# Add higher in somatic scRNAseq data of normal tissues from the Human Protein Atlas
all_genes <- all_genes %>%
left_join(as_tibble(rowData(scRNAseq_HPA), rownames = "ensembl_gene_id") %>%
dplyr::select(ensembl_gene_id, external_gene_name,
Higher_in_somatic_cell_type))
# CCLE database analysis added
all_genes <- all_genes %>%
left_join(as_tibble(rowData(CCLE_data), rownames = "ensembl_gene_id"))
all_genes[is.na(all_genes$CCLE_category), "CCLE_category"] <- "not_in_CCLE"
# TCGA database analysis added
all_genes <- all_genes %>%
left_join(as_tibble(rowData(TCGA_TPM), rownames = "ensembl_gene_id") %>%
dplyr::select(ensembl_gene_id, external_gene_name, percent_pos_tum,
percent_neg_tum, max_TPM_in_TCGA, TCGA_category))
all_genes[all_genes$lowly_expressed_in_GTEX == TRUE &
all_genes$multimapping_analysis == "testis_specific",
"TCGA_category"] <- "multimapping_issue"
# Add DAC analysis
induced <- as_tibble(rowData(DAC_treated_cells_multimapping),
rownames = "ensembl_gene_id") %>%
filter(induced == TRUE) %>%
pull(external_gene_name)
all_genes <- all_genes %>%
mutate(DAC_induced = case_when(external_gene_name %in% induced ~ TRUE,
!external_gene_name %in% induced ~ FALSE))
# Add the most biologically relevant transcript (canonical and coordinates)
ensembl <- biomaRt::useDataset("hsapiens_gene_ensembl", useMart("ensembl"))
attributes_vector <- c("ensembl_gene_id",
"external_gene_name",
"ensembl_transcript_id",
"external_transcript_name",
"chromosome_name",
"strand",
"transcript_start",
"transcript_end",
"transcription_start_site",
"transcript_length",
"transcript_biotype",
"transcript_is_canonical")
transcripts_infos <- as_tibble(biomaRt::getBM(attributes = attributes_vector,
mart = ensembl))
canonical_transcripts <- transcripts_infos %>%
filter(transcript_is_canonical == 1) %>%
filter(chromosome_name %in% c(1:22, "X", "Y", "MT")) %>%
filter(transcript_biotype == "protein_coding" | transcript_biotype == "lncRNA")
all_genes <- all_genes %>%
left_join(canonical_transcripts %>%
dplyr::select(ensembl_gene_id,
external_transcript_name, ensembl_transcript_id,
chromosome_name, strand, transcript_start,
transcript_end, transcription_start_site,
transcript_length, transcript_biotype))From there, we filtered based on the testis_specificity (“testis_specific” or “testis_preferential”), testis_cell_type (not “Macrophage”, “Endothelial”, “Myoid”, “Sertoli”, “Leydig”), Higher_in_somatic_cell_type (TRUE), CCLE_category (“activated”) and TCGA_category (“activated” or “multimapping_issue”) to have our CT genes. Then the most relevant transcript was validated manually (and check that there is a transcript activated in testis and the same is activated in tumor).
2.3 CTexploreR VS CTdatabase
CTdatabase_ours <- Venn(list(CTdatabase = CTdatabase_cleaned$external_gene_name,
CTexploreR = CT_genes$external_gene_name))
gp <- VennThemes(compute.Venn(CTdatabase_ours))
gp[["Face"]][["11"]]$fill <- "mistyrose"
gp[["Face"]][["01"]]$fill <- "darkseagreen1"
gp[["Face"]][["10"]]$fill <- "lightsteelblue1"
gp[["Set"]][["Set1"]]$col <- "paleturquoise4"
gp[["Set"]][["Set2"]]$col <- "darkseagreen4"
gp[["SetText"]][["Set1"]]$col <- "paleturquoise4"
gp[["SetText"]][["Set2"]]$col <- "darkseagreen4"
plot(CTdatabase_ours, gp = gp)
Lost genes analysis
CTdatabase_lost <- all_genes %>%
filter(external_gene_name %in% CTdatabase_ours@IntersectionSets[["10"]])
# 10 Genes are lost because not in any database
CTdatabase_lost[is.na(CTdatabase_lost$testis_specificity), ]$testis_specificity <- "not_specific"
table(CTdatabase_lost$testis_specificity)##
## not_specific testis_preferential testis_specific
## 85 14 38table(CTdatabase_lost$testis_cell_type)##
## Early_spermatocyte Elongated_spermatid Late_spermatocyte Leydig
## 15 14 15 2
## Round_spermatid Sertoli Sperm1 Sperm2
## 39 2 5 9
## Spermatogonia SSC
## 2 11table(CTdatabase_lost$Higher_in_somatic_cell_type)##
## FALSE TRUE
## 95 28table(CTdatabase_lost$TCGA_category)##
## activated leaky multimapping_issue not_activated
## 48 46 7 36table(CTdatabase_lost$CCLE_category)##
## activated leaky not_activated
## 54 32 512.4 CTexploreR VS omics databases
core_ours <- Venn(list(Jamin = Jamin_core_CT$Gene,
CTexploreR = CT_genes$external_gene_name))
Wang_ours <- Venn(list(Wang = Wang_CT$external_gene_name,
CTexploreR = CT_genes$external_gene_name))
Carter_ours <- Venn(list(Carter_CT = Carter_CT$Gene_Name,
CTexploreR = CT_genes$external_gene_name))
gene_list <- list(Jamin = Jamin_core_CT$Gene,
CTdatabase = CTdatabase_cleaned$external_gene_name,
CTatlas = Wang_CT$external_gene_name,
CTexploreR = CT_genes$external_gene_name,
Carter = Carter_CT$Gene_Name)
upset_omics <- fromList(gene_list[-2])
upset(upset_omics)
Lost genes analysis
Jamin_lost <- all_genes %>%
filter(external_gene_name %in% core_ours@IntersectionSets[["10"]])
Jamin_lost[is.na(Jamin_lost$testis_specificity), ]$testis_specificity <- "not_specific"
table(Jamin_lost$testis_specificity)##
## not_specific testis_preferential testis_specific
## 78 5 2table(Jamin_lost$testis_cell_type)##
## Early_spermatocyte Elongated_spermatid Endothelial Late_spermatocyte
## 10 9 1 13
## Macrophage Round_spermatid Sertoli Sperm1
## 1 22 2 4
## Sperm2 Spermatogonia SSC
## 4 4 4table(Jamin_lost$Higher_in_somatic_cell_type)##
## FALSE TRUE
## 60 22table(Jamin_lost$TCGA_category)##
## activated leaky not_activated
## 15 56 14table(Jamin_lost$CCLE_category)##
## activated leaky not_activated
## 39 42 4Wang_lost <- all_genes %>%
filter(external_gene_name %in% Wang_ours@IntersectionSets[["10"]])
Wang_lost[is.na(Wang_lost$testis_specificity), ]$testis_specificity <- "not_specific"
table(Wang_lost$testis_specificity)##
## not_specific testis_preferential testis_specific
## 470 109 249table(Wang_lost$testis_cell_type)##
## Early_spermatocyte Elongated_spermatid Endothelial Late_spermatocyte
## 69 137 1 76
## Leydig Myoid Round_spermatid Sertoli
## 1 1 308 18
## Sperm1 Sperm2 Spermatogonia SSC
## 41 69 22 43table(Wang_lost$Higher_in_somatic_cell_type)##
## FALSE TRUE
## 734 66table(Wang_lost$TCGA_category)##
## activated leaky multimapping_issue not_activated
## 302 302 5 219table(Wang_lost$CCLE_category)##
## activated leaky not_activated
## 256 206 366Carter_lost <- all_genes %>%
filter(external_gene_name %in% Carter_ours@IntersectionSets[["10"]])
Carter_lost[is.na(Carter_lost$testis_specificity), ]$testis_specificity <- "not_specific"
table(Carter_lost$testis_specificity)##
## not_specific testis_preferential testis_specific
## 1 8 42table(Carter_lost$testis_cell_type)##
## Early_spermatocyte Elongated_spermatid Late_spermatocyte Round_spermatid
## 4 16 3 20
## Sertoli Sperm1 Spermatogonia SSC
## 2 1 1 2table(Carter_lost$Higher_in_somatic_cell_type)##
## FALSE TRUE
## 48 3table(Carter_lost$TCGA_category)##
## activated leaky not_activated
## 23 9 19table(Carter_lost$CCLE_category)##
## activated leaky not_activated
## 10 3 382.5 Characterisation of differences with all databases
common <- unique(c(core_ours@IntersectionSets[["11"]],
CTdatabase_ours@IntersectionSets[["11"]],
Wang_ours@IntersectionSets[["11"]],
Carter_ours@IntersectionSets[["11"]]))
length(common)## [1] 212length(common)/dim(CT_genes)[1] * 100## [1] 71.14094lost_list <- unique(c(core_ours@IntersectionSets[["10"]],
CTdatabase_ours@IntersectionSets[["10"]],
Wang_ours@IntersectionSets[["10"]],
Carter_ours@IntersectionSets[["10"]]))
lost <- all_genes %>%
filter(external_gene_name %in% lost_list)
not_specific <- filter(lost, is.na(testis_specificity))
GTEX_expression(not_specific$external_gene_name, units = "log_TPM")
somatic_testis <- filter(lost, testis_cell_type %in% c( "Macrophage",
"Endothelial", "Myoid",
"Sertoli", "Leydig"))
testis_expression(somatic_testis$external_gene_name, cells = "all")## `use_raster` is automatically set to TRUE for a matrix with more than
## 2000 columns You can control `use_raster` argument by explicitly
## setting TRUE/FALSE to it.
##
## Set `ht_opt$message = FALSE` to turn off this message.
somatic_tissue <- filter(lost, Higher_in_somatic_cell_type == TRUE)
HPA_cell_type_expression(somatic_tissue$external_gene_name)
not_TCGA_activated <- filter(lost, TCGA_category != "activated" &
TCGA_category != "multimapping_issue")
TCGA_expression(not_TCGA_activated$external_gene_name,
tumor = "all",
units = "log_TPM")## `use_raster` is automatically set to TRUE for a matrix with more than
## 2000 columns You can control `use_raster` argument by explicitly
## setting TRUE/FALSE to it.
##
## Set `ht_opt$message = FALSE` to turn off this message.
not_CCLE_activated <- filter(lost, CCLE_category != "activated")
CCLE_expression(not_CCLE_activated$external_gene_name,
type = c("lung", "skin", "colorectal",
"gastric", "breast", "head_and_neck"),
units = "log_TPM")
transcript_prob <- lost %>%
filter(testis_specificity == "testis_specific" |
testis_specificity == "testis_preferential") %>%
filter(TCGA_category == "activated" | TCGA_category == "multimapping_issue") %>%
filter(CCLE_category == "activated") %>%
dim()We have lost 979 genes in total. Among them, 58.0183861% are not considered testis specific, 2.8600613% are expressed in testis somatic cells, 11.13381% are expressed in somatic cells, 62.206333% are not activated in TCGA samples, 66.4964249% are not activated in CCLE cell lines and 1.9407559% is lost due to transcripts problems.
What about new genes in CTexploreR
new <- CT_genes %>%
filter(!external_gene_name%in%common)
newtable(new$testis_specificity)##
## testis_preferential testis_specific
## 24 62table(new$X_linked, new$regulated_by_methylation)##
## FALSE TRUE
## FALSE 50 24
## TRUE 1 11TCGA_expression(tumor = "all", genes = new$external_gene_name,
units = "log_TPM")## `use_raster` is automatically set to TRUE for a matrix with more than
## 2000 columns You can control `use_raster` argument by explicitly
## setting TRUE/FALSE to it.
##
## Set `ht_opt$message = FALSE` to turn off this message.
TCGA_expression(tumor = "all",
genes = filter(new, X_linked & regulated_by_methylation)$external_gene_name,
units = "log_TPM")## `use_raster` is automatically set to TRUE for a matrix with more than
## 2000 columns You can control `use_raster` argument by explicitly
## setting TRUE/FALSE to it.
##
## Set `ht_opt$message = FALSE` to turn off this message.
There are 86 new CT genes in CTexploreR. These are mainly testis
specific and on autosomes. Regulation by methylation is not the majority
of them. This makes sens as other databases have been focused on
regulation by methylation, so as here we don’t segregate with that we
can find these. There is only 11 new “major” CT that are on the X
chromosome and regulated by methylation. CT45 are not that new.
Expression in tumours doesn’t strike that much.
3 CT genes characteristics
table(CT_genes$testis_specificity)##
## testis_preferential testis_specific
## 92 206table(CT_genes$transcript_biotype)##
## lncRNA nonsense_mediated_decay protein_coding
## 48 3 247Most genes are testis specific (69.1275168%). Most genes are mainly protein coding genes (82.885906%).
3.1 X chromosome and regulated by methylation
In CTexploreR, genes have been characterised as regulated by methylation or not.
table(CT_genes$X_linked)##
## FALSE TRUE
## 205 93table(CT_genes$regulated_by_methylation)##
## FALSE TRUE
## 138 160table(CT_genes$X_linked, CT_genes$testis_specificity)##
## testis_preferential testis_specific
## FALSE 86 119
## TRUE 6 87table(CT_genes$X_linked, CT_genes$regulated_by_methylation)##
## FALSE TRUE
## FALSE 128 77
## TRUE 10 83Genes are enriched on the X chromosome (31.2080537%). Also, 160 genes have been flagged as regulated by methylation (53.6912752%). It is interesting to study the link between these two characteristics.
On the chromosome X, there is a clear enrichment of CT genes regulated by methylation (83/93 chrX genes or 83/160 genes regulated by methylation).
Let’s check that with a statistical test, I here want to see if, on there is an enrichment of genes regulated by methylation on the X chromosome. I need to do a Pearson Chi square test (to know if observed proportion differ from expected proportion). It is a statistical method to determine if two categorical variables have a significant correlation between them. I can directly put a matrix (my table) in the function
chisq.test(table(CT_genes$X_linked, CT_genes$regulated_by_methylation))##
## Pearson's Chi-squared test with Yates' continuity correction
##
## data: table(CT_genes$X_linked, CT_genes$regulated_by_methylation)
## X-squared = 66.676, df = 1, p-value = 3.2e-16CT_genes$chr_factor <- factor(CT_genes$chr,
levels = c("1", "2", "3", "4", "5", "6", "7", "8", "9",
"10", "11", "12", "13", "14", "15", "16", "17",
"18", "19", "20", "21", "22", "X", "Y"))
CT_genes %>%
mutate(regulated_by_methylation = ifelse(regulated_by_methylation,
"Regulated by methylation",
"Not regulated by methylation")) %>%
ggplot(aes(x = chr_factor, fill = testis_specificity, color = X_linked)) +
geom_bar(stat = 'count') +
scale_fill_manual(values = c("goldenrod1", "mediumpurple1")) +
scale_color_manual(values = c("royalblue1", "deeppink"), guide = "none") +
facet_wrap(~ regulated_by_methylation, ncol = 2) +
theme_bw() +
xlab("Chromosome") +
ylab("Number of genes") +
theme(axis.text.x = element_text(angle = 90, hjust = 1),
legend.position = "top",
axis.title = element_text(size = 10, color = "gray10"))
There is indeed a significative link between regulation by methylation and being on the X chromosome. There is thus an enrichment of CT genes regulated by methylation on the X chromosome (and inversely).
3.2 Tumour and methylation
Using CTexploreR functions, we can explore all CT genes or focus on some potential targets.
3.2.1 All CT
For these heatmaps, the code comes from the function but has been copies to add some annotations.
chr_mat <- as.matrix(CT_genes$X_linked)
chr_mat <- ifelse(chr_mat == TRUE, "X-linked", "Not X")
rownames(chr_mat) <- CT_genes$external_gene_name
row_ha_chr <- rowAnnotation(chr_factor = chr_mat,
annotation_legend_param = legends_param,
simple_anno_size = unit(0.5, "cm"),
col = list(chr_factor = chr_colors),
annotation_name_gp = gpar(fontsize = 8),
annotation_name_side = "top")
regulation_mat <- as.matrix(CT_genes$regulated_by_methylation)
regulation_mat <- ifelse(regulation_mat == TRUE, "Methylation",
"Not methylation")
rownames(regulation_mat) <- CT_genes$external_gene_name
row_ha_reg <- rowAnnotation(regulation = regulation_mat,
annotation_legend_param = legends_param,
simple_anno_size = unit(0.5, "cm"),
col = list(regulation = meth_colors),
annotation_name_gp = gpar(fontsize = 8),
annotation_name_side = "top")
left_annot <- c(row_ha_chr, row_ha_reg, gap = unit(1, "mm"))
split <- data.frame(CT_genes$regulated_by_methylation, CT_genes$X_linked)database <- TCGA_TPM
database$tumor <- sub(pattern = 'TCGA-', x = database$project_id, '')
database$type <- "Tumor"
database$type[database$shortLetterCode == "NT"] <- "Peritumoral"
database <- database[, order(database$tumor, database$type)]
genes <- CT_genes$external_gene_name
database <- database[rowData(database)$external_gene_name %in% genes, ]
database <- database[match(genes, rowData(database)$external_gene_name), ]
database <- database[, database$type == "Tumor"]
column_ha_tumor <- HeatmapAnnotation(
Tumor = database$tumor,
border = TRUE,
col = list(Tumor = c("BRCA" = "midnightblue", "COAD" = "darkorchid2",
"ESCA" = "gold", "HNSC" = "deeppink2",
"LUAD" = "seagreen", "LUSC" = "seagreen3",
"SKCM" = "red3")),
annotation_name_gp = gpar(fontsize = 8),
annotation_legend_param = legends_param)
split_by <- factor(database$tumor)
col_annot <- column_ha_tumor
mat <- log1p(assay(database))
rownames(mat) <- rowData(database)$external_gene_name
Heatmap(mat[, , drop = FALSE],
name = "logTPM",
column_title = paste0("Expression in TCGA samples (all)"),
column_split = split_by,
row_split = split,
row_title_gp = gpar(fontsize = 0),
col = colorRamp2(seq(0, max(mat), length = 11),
legend_colors),
clustering_method_rows = "ward.D",
clustering_method_columns = "ward.D",
cluster_rows = TRUE,
show_column_names = FALSE,
cluster_columns = TRUE,
show_column_dend = FALSE,
show_row_dend = FALSE,
row_names_gp = gpar(fontsize = 4),
heatmap_legend_param = legends_param,
top_annotation = col_annot,
left_annotation = left_annot)
database <- CCLE_data
database$type <- tolower(database$type)
genes <- CT_genes$external_gene_name
database <- database[rowData(database)$external_gene_name %in% genes, ]
database <- database[match(genes, rowData(database)$external_gene_name), ]
mat <- log1p(assay(database))
rownames(mat) <- rowData(database)$external_gene_name
df_col <- data.frame("cell_line" = colData(database)$cell_line_name,
"type" = as.factor(colData(database)$type))
rownames(df_col) <- rownames(colData(database))
df_col <- df_col[order(df_col$type), ]
column_ha_type <- HeatmapAnnotation(
type = df_col$type,
border = TRUE,
annotation_name_gp = gpar(fontsize = 8),
annotation_legend_param = legends_param,
col = list(type = c("lung" = "seagreen3", "skin" = "red3",
"bile_duct" = "mediumpurple1", "bladder" = "mistyrose2",
"colorectal" = "plum", "lymphoma" = "steelblue1",
"uterine" = "darkorange4", "myeloma" = "turquoise3",
"kidney" = "thistle4",
"pancreatic" = "darkmagenta", "brain" = "palegreen2",
"gastric" = "wheat3", "breast" = "midnightblue",
"bone" = "sienna1", "head_and_neck" = "deeppink2",
"ovarian" = "tan3", "sarcoma" = "lightcoral",
"leukemia" = "steelblue4", "esophageal"= "khaki",
"neuroblastoma" = "olivedrab1")))
Heatmap(mat[, rownames(df_col), drop = FALSE],
name = "logTPM",
column_title = "Gene Expression in tumor cell lines (CCLE)",
column_split = factor(df_col$type),
row_split = split,
row_title_gp = gpar(fontsize = 0),
col = colorRamp2(seq(0, max(mat), length = 11),
legend_colors),
clustering_method_rows = "ward.D",
clustering_method_columns = "ward.D",
cluster_rows = TRUE,
show_row_dend = FALSE,
show_column_names = FALSE,
cluster_columns = TRUE,
show_column_dend = FALSE,
row_names_gp = gpar(fontsize = 4),
heatmap_legend_param = legends_param,
top_annotation = c(column_ha_type),
left_annotation = left_annot)
Next graph was removed from paper
genes <- CT_genes$external_gene_name
database <- CT_mean_methylation_in_tissues[rownames(CT_mean_methylation_in_tissues) %in% genes]
mat <- na.omit(assay(database))
clustering_option <- TRUE
row_ha_chr_meth <- rowAnnotation(chr = chr_mat[rownames(mat),],
annotation_legend_param = legends_param,
simple_anno_size = unit(0.5, "cm"),
col = list(chr = chr_colors),
annotation_name_gp = gpar(fontsize = 8),
annotation_name_side = "top")
row_ha_reg_meth <- rowAnnotation(regulation = regulation_mat[rownames(mat),],
annotation_legend_param = legends_param,
simple_anno_size = unit(0.5, "cm"),
col = list(regulation = meth_colors),
annotation_name_gp = gpar(fontsize = 8),
annotation_name_side = "top")
left_annot_meth <- c(row_ha_chr_meth, row_ha_reg_meth, gap = unit(1, "mm"))
split_meth <- data.frame(filter(CT_genes, external_gene_name %in% rownames(mat))$regulated_by_methylation,
filter(CT_genes, external_gene_name %in% rownames(mat))$X_linked)
Heatmap(mat,
column_title = 'Promoter mean methylation level by tissue',
name = 'Meth',
col = colorRamp2(c(1:100),
colorRampPalette(c("moccasin","dodgerblue4"))(100)),
na_col = "gray80",
cluster_rows = clustering_option,
cluster_columns = FALSE,
row_split = split_meth,
row_title_gp = gpar(fontsize = 0),
show_row_names = TRUE,
show_heatmap_legend = TRUE,
show_row_dend = FALSE,
row_names_gp = gpar(fontsize = 3),
column_names_gp = gpar(fontsize = 8),
column_names_side = "bottom",
row_names_side = "right",
left_annotation = left_annot_meth)
3.2.2 Coactivation in tumours
We know that CT genes that are regulated by methylation are activated in tumours due to methylation. However, those that are not it is more blurry. It is interesting to look at their expression to see if there are groups of genes that are activated together in tumours which would suggest another activation : a transcription factor ? a gene expresion program from meiosis ? timing of expression ? function ?
not_regulated <- filter(CT_genes, !regulated_by_methylation)$external_gene_nameAs they can blur the clustering, I sometimes remove genes that are in the background and those that are expressed a lot.
- BRCA :
TCGA_expression(filter(CT_genes, !regulated_by_methylation)$external_gene_name,
tumor = "BRCA", units = "log_TPM")
# Removing genes to see clearer
BRCA_genes <- rowData(TCGA_TPM) %>%
as_tibble() %>%
filter(external_gene_name %in% not_regulated) %>%
filter(percent_pos_BRCA > 0.01 & percent_pos_BRCA < 30) %>%
pull(external_gene_name)
BRCA_genes_ordered <-
BRCA_genes[row_order(draw(TCGA_expression(BRCA_genes, tumor = "BRCA",
units = "log_TPM")))]
BRCA_cluster1 <- BRCA_genes_ordered[c(which(BRCA_genes_ordered == "AKAP14"):
which(BRCA_genes_ordered == "C11orf97"))]
BRCA_cluster2 <- BRCA_genes_ordered[c(which(BRCA_genes_ordered == "GTF2I-AS1"):
which(BRCA_genes_ordered == "SLIT3-AS1"))]We can see two small clusters.
- LUAD :
TCGA_expression(filter(CT_genes, !regulated_by_methylation)$external_gene_name,
tumor = "LUAD", units = "log_TPM")
# Removing genes to see clearer
LUAD_genes <- rowData(TCGA_TPM) %>%
as_tibble() %>%
filter(external_gene_name %in% not_regulated) %>%
filter(percent_pos_LUAD > 0.01 & percent_pos_LUAD < 30) %>%
pull(external_gene_name)
LUAD_genes_ordered <-
LUAD_genes[row_order(draw(TCGA_expression(LUAD_genes, tumor = "LUAD",
units = "log_TPM")))]
LUAD_cluster1 <- c("TSPY1", "TSPY2")
LUAD_cluster2 <- c("SPANXC", "SPANXB1", "SPANXD")
LUAD_cluster3 <- LUAD_genes_ordered[c(1:22)]
LUAD_cluster4 <- LUAD_genes_ordered[c(which(LUAD_genes_ordered == "GTF2I-AS1"):
which(LUAD_genes_ordered == "SLIT3-AS1"))]22 first genes are clearly quite clustering, there is also a small SPANX cluster, a small TSPY cluster ans a bigger one around the middle of the graph.
- LUSC :
TCGA_expression(filter(CT_genes, !regulated_by_methylation)$external_gene_name,
tumor = "LUSC", units = "log_TPM")
# Removing genes to see clearer
LUSC_genes <- rowData(TCGA_TPM) %>%
as_tibble() %>%
filter(external_gene_name %in% not_regulated) %>%
filter(percent_pos_LUSC > 0.01 & percent_pos_LUSC < 30) %>%
pull(external_gene_name)
LUSC_genes_ordered <-
LUSC_genes[row_order(draw(TCGA_expression(LUSC_genes, tumor = "LUSC",
units = "log_TPM")))]
LUSC_cluster1 <- c("TSPY1", "TSPY2", "TSPY9", "TSPY10")
LUSC_cluster2 <- c("SPANXC", "SPANXB1", "SPANXD")
LUSC_cluster3 <- LUSC_genes_ordered[c(1:15)]We can see a cluster at the top and two smaller ones corresponding to SPANX and TSPY genes. As well as quite a big cluster with the 15 first lines
- SKCM :
SKCM_genes <- rowData(TCGA_TPM) %>%
as_tibble() %>%
filter(external_gene_name %in% not_regulated) %>%
filter(percent_pos_SKCM > 0.01 & percent_pos_SKCM < 30) %>%
pull(external_gene_name)
TCGA_expression(filter(CT_genes, !regulated_by_methylation)$external_gene_name,
tumor = "SKCM", units = "log_TPM")
# Lots of genes activated almost everywhere or nowhere that can mask clusters
TCGA_expression(SKCM_genes,
tumor = "SKCM", units = "log_TPM")
SKCM_cluster <- c("TSPY1", "TSPY2", "TSPY3", "TSPY9", "TSPY10")It is clear that there is a TSPY cluster again.
- Comparison
TSPY and SPANX genes are found reactivated/coactivated in different types of tumours. Let’s compare the bigger groups found in LUAD, LUSC and BRCA
upset(fromList(list(LUSC = LUSC_cluster3,
LUAD1 = LUAD_cluster3,
LUAD2 = LUAD_cluster4,
BRCA1 = BRCA_cluster1,
BRCA2 = BRCA_cluster2)))
There is quite a lot of similarities here between the clusters. Let’s look at the expression in tumours.
TCGA_expression(c(LUSC_cluster3, LUAD_cluster3, LUAD_cluster4, BRCA_cluster1,
BRCA_cluster2),
tumor = "all", units = "log_TPM")## `use_raster` is automatically set to TRUE for a matrix with more than
## 2000 columns You can control `use_raster` argument by explicitly
## setting TRUE/FALSE to it.
##
## Set `ht_opt$message = FALSE` to turn off this message.
Let’s have a look at the timings of expression.
testis_expression(c("TSPY1", "TSPY2", "TSPY3", "TSPY9", "TSPY10"),
cells = "all")
testis_expression(c("SPANXC", "SPANXB1", "SPANXD"),
cells = "all")
testis_expression(LUSC_cluster3, cells = "all")
testis_expression(LUAD_cluster3, cells = "all")
testis_expression(LUAD_cluster4, cells = "all")## Warning: 4 out of 6 names invalid: GTF2I-AS1, NFIA-AS1, CCDC196, SLIT3-AS1.
## See the manual page for valid types.
testis_expression(BRCA_cluster1, cells = "all")
testis_expression(BRCA_cluster2, cells = "all")## Warning: 3 out of 8 names invalid: GTF2I-AS1, NFIA-AS1, SLIT3-AS1.
## See the manual page for valid types.
They mainly seem to, in a cluster, be expressed at the same time.
3.2.3 MAGE genes
MAGE_genes <- filter(CT_genes, family == "MAGE")$external_gene_name
TCGA_expression(tumor = "all",
genes = MAGE_genes,
units = "log_TPM")## `use_raster` is automatically set to TRUE for a matrix with more than
## 2000 columns You can control `use_raster` argument by explicitly
## setting TRUE/FALSE to it.
##
## Set `ht_opt$message = FALSE` to turn off this message.
CCLE_expression(genes = MAGE_genes,
type = c("lung", "skin", "bile_duct", "bladder",
"colorectal", "lymphoma", "uterine",
"myeloma", "kidney", "pancreatic", "brain",
"gastric", "breast", "bone", "head_and_neck",
"ovarian", "sarcoma", "leukemia", "esophageal",
"neuroblastoma"), units = "log_TPM")
normal_tissues_mean_methylation(MAGE_genes, na.omit = TRUE)
3.3 Single Cell analysis : timing of expression
In CTexploreR, we there is single cell data from the testis, we thus can analyse CT genes expression during spermatogenesis.
There is only data for 251 of our 298 CT genes.
NB : SSC, Spermatogonia and early spermatocytes are premeiotic cells. Late spermatocytes (between both meiosis), round spermatid, elongated spermatid and sperm are postmeiotic cells.
genes_avail <-
CT_genes$external_gene_name[CT_genes$external_gene_name %in% unique(rownames(testis_sce))]
table(CT_genes$testis_cell_type)##
## Early_spermatocyte Elongated_spermatid Late_spermatocyte Round_spermatid
## 41 25 23 64
## Sperm1 Sperm2 Spermatogonia SSC
## 12 14 29 33table(CT_genes$testis_cell_type)/length(genes_avail)*100##
## Early_spermatocyte Elongated_spermatid Late_spermatocyte Round_spermatid
## 16.334661 9.960159 9.163347 25.498008
## Sperm1 Sperm2 Spermatogonia SSC
## 4.780876 5.577689 11.553785 13.14741041% of genes are mainly expressed pre-meioticly.
germ_cells <- c("SSC", "Spermatogonia", "Early_spermatocyte",
"Late_spermatocyte","Round_spermatid", "Elongated_spermatid",
"Sperm1", "Sperm2")
somatic_cells <- c("Macrophage", "Endothelial", "Myoid", "Sertoli", "Leydig")
testis_sce_CT <- testis_sce[genes_avail, ]
mat <- SingleCellExperiment::logcounts(testis_sce_CT)
df_col <- data.frame(clusters = colData(testis_sce_CT)$clusters,
type = colData(testis_sce_CT)$type,
Donor = colData(testis_sce_CT)$Donor)
rownames(df_col) <- colnames(testis_sce_CT)
df_col <- df_col[order(df_col$type),]
df_col$lineage <- "Germ cells"
df_col$lineage[df_col$type %in% somatic_cells] <- "Somatic cells"
column_ha_type = HeatmapAnnotation(
type = df_col$type,
border = TRUE,
col = list(type = c("SSC" = "floralwhite", "Spermatogonia" = "moccasin",
"Early_spermatocyte" = "gold",
"Late_spermatocyte" = "orange",
"Round_spermatid" = "red2",
"Elongated_spermatid" = "darkred",
"Sperm1" = "violet", "Sperm2" = "purple",
"Sertoli" = "gray",
"Leydig" = "cadetblue2", "Myoid" = "springgreen3",
"Macrophage" = "gray10",
"Endothelial" = "steelblue")),
annotation_name_gp = gpar(fontsize = 8),
annotation_legend_param = legends_param)
column_ha_lineage = HeatmapAnnotation(
lineage = df_col$lineage,
border = TRUE,
col = list(lineage = c("Germ cells" = "salmon", "Somatic cells" = "cyan4")),
annotation_name_gp = gpar(fontsize = 8),
annotation_legend_param = legends_param)
scale_lims <- c(0, quantile(rowMax(mat), 0.75))
top_annot <- c(column_ha_lineage, column_ha_type)
# Until here is what's in the function, hereunder is my addition/change in Heatmap()
CT_genes_avail <- filter(CT_genes, external_gene_name %in% genes_avail)
chr_mat <- as.matrix(CT_genes_avail$X_linked)
chr_mat <- ifelse(chr_mat == TRUE, "X-linked", "Not X")
rownames(chr_mat) <- CT_genes_avail$external_gene_name
row_ha_chr <- rowAnnotation(chr = chr_mat,
annotation_legend_param = legends_param,
simple_anno_size = unit(0.5, "cm"),
col = list(chr = chr_colors),
annotation_name_gp = gpar(fontsize = 8),
annotation_name_side = "top")
regulation_mat <- as.matrix(CT_genes_avail$regulated_by_methylation)
regulation_mat <- ifelse(regulation_mat == TRUE, "Methylation",
"Not methylation")
rownames(regulation_mat) <- CT_genes_avail$external_gene_name
row_ha_reg <- rowAnnotation(regulation = regulation_mat,
annotation_legend_param = legends_param,
simple_anno_size = unit(0.5, "cm"),
col = list(regulation = meth_colors),
annotation_name_gp = gpar(fontsize = 8),
annotation_name_side = "top")
left_annot <- c(row_ha_chr, row_ha_reg, gap = unit(1, "mm"))
split <- data.frame(CT_genes_avail$regulated_by_methylation, CT_genes_avail$X_linked)
Heatmap(mat[genes_avail, rownames(df_col), drop = FALSE],
name = "logCounts",
column_title = "Expression in testis cells (scRNAseq)",
column_split = df_col$type,
row_split = split,
row_title_gp = gpar(fontsize = 0),
show_column_names = FALSE,
show_column_dend = FALSE,
clustering_method_rows = "ward.D",
clustering_method_columns = "ward.D",
cluster_rows = TRUE,
cluster_columns = FALSE,
show_row_dend = FALSE,
row_names_gp = gpar(fontsize = 4),
col = colorRamp2(seq(scale_lims[1], scale_lims[2], length = 11),
legend_colors),
top_annotation = top_annot,
left_annotation = left_annot,
heatmap_legend_param = legends_param)
We used these data to determine in which testis cell type each gene is mostly expressed.
CT_genes$main_cell_type_expression <- factor(CT_genes$testis_cell_type,
levels = c("SSC",
"Spermatogonia",
"Early_spermatocyte",
"Late_spermatocyte",
"Round_spermatid",
"Elongated_spermatid",
"Sperm1",
"Sperm2",
"Sertoli"))
CT_genes %>%
mutate(regulated_by_methylation = ifelse(regulated_by_methylation,
"Regulated by methylation",
"Not regulated by methylation")) %>%
filter(!is.na(testis_cell_type)) %>%
filter(main_cell_type_expression != "Sertoli") %>%
ggplot(aes(x = chr, fill = main_cell_type_expression,
color = main_cell_type_expression)) +
scale_fill_manual(values = c("lightyellow2", "moccasin", "gold", "orange","red2",
"darkred", "violet", "purple")) +
scale_color_manual(values = c("lightyellow3", "navajowhite2", "gold", "orange","red2",
"darkred", "violet", "purple")) +
geom_bar(stat = 'count') +
facet_grid(main_cell_type_expression ~ regulated_by_methylation) +
xlab("Chromosome") +
ylab("Number of genes") +
theme_bw() +
theme(
axis.text.x = element_text(color = "black", angle = 90, vjust = 0.5, size = 10),
axis.title = element_text(size = 10, color = "gray10"),
strip.text.y = element_blank()) 
14 genes are on the X chromosome but escape X chromosome inactivation and are activates post-meioticly.
CT_genes %>%
filter(X_linked) %>%
filter(testis_cell_type %in% c("Late_spermatocyte","Round_spermatid",
"Elongated_spermatid", "Sperm1", "Sperm2"))3.4 Gene function
All CT genes function
msigdbr(species = "Homo sapiens" , category = "C5") %>%
filter(gene_symbol %in% CT_genes$external_gene_name) %>%
pull(gene_symbol) %>%
unique() %>%
length()## [1] 184msigdbr(species = "Homo sapiens" , category = "H") %>%
filter(gene_symbol %in% CT_genes$external_gene_name) %>%
pull(gene_symbol) %>%
unique() %>%
length()## [1] 17go_ora <- enrichGO(gene = CT_genes$ensembl_gene_id,
keyType = "ENSEMBL",
OrgDb = org.Hs.eg.db,
ont = "all",
readable = TRUE)
as_tibble(go_ora)as_tibble(go_ora) %>%
arrange(desc(Count)) %>%
head(12) %>%
mutate(Ratio = case_when(ONTOLOGY == "BP"~ Count/177,
ONTOLOGY == "CC"~ Count/197,
ONTOLOGY == "MF"~ Count/186)) %>%
ggplot(aes(x = Ratio, y = Description, fill = Description)) +
geom_col() +
theme_bw() +
ylab("GO term") +
xlab("Gene Ratio") +
theme(axis.text.y = element_blank(),
legend.position = "none",
axis.ticks.y = element_blank(),
axis.title = element_text(size = 10, color = "gray10"))+
geom_text(aes(0, y = Description, label = Description),
hjust = 0,
nudge_x = 0.005,
colour = "floralwhite",
size = 4)
ora_to_plot <- as_tibble(simplify(go_ora))
# Combine the two cilium movement from BP
ora_to_plot[ora_to_plot$ID == "GO:0003341", "Count"] <-
ora_to_plot[ora_to_plot$ID == "GO:0003341", "Count"] +
ora_to_plot[ora_to_plot$ID == "GO:0060294", "Count"]
ora_to_plot <- ora_to_plot %>%
arrange(desc(Count)) %>%
head(11) %>%
filter(ID != "GO:0060294") %>%
mutate(Ratio = case_when(ONTOLOGY == "BP"~ Count/177,
ONTOLOGY == "CC"~ Count/197,
ONTOLOGY == "MF"~ Count/186))
ora_to_plot %>%
ggplot(aes(x = Ratio, y = Description, fill = Description)) +
geom_col() +
theme_bw() +
ylab("GO term") +
xlab("Gene Ratio") +
theme(axis.text.y = element_blank(),
legend.position = "none",
axis.ticks.y = element_blank(),
axis.title = element_text(size = 10, color = "gray10"))+
geom_text(aes(0, y = Description, label = Description),
hjust = 0,
nudge_x = 0.005,
colour = "floralwhite",
size = 3.7)
As we can see here, most of genes are indeed linked to functions from reproduction. I represented here the 12 categories with the most genes, all from biological processes. However, they are enriched in 89 different GO terms.
Is there a difference between meth reg or not ?
go_ora_meth <- enrichGO(gene =
filter(CT_genes, regulated_by_methylation)$ensembl_gene_id,
keyType = "ENSEMBL",
OrgDb = org.Hs.eg.db,
ont = "all",
readable = TRUE)
go_ora_meth <- simplify(go_ora_meth)
go_ora_not_meth <- enrichGO(gene =
filter(CT_genes, !regulated_by_methylation)$ensembl_gene_id,
keyType = "ENSEMBL",
OrgDb = org.Hs.eg.db,
ont = "all",
readable = TRUE)
go_ora_not_meth <- simplify(go_ora_not_meth)
as_tibble(go_ora_not_meth)go_ora_meth_plot <- as_tibble(go_ora_meth) %>%
arrange(desc(Count)) %>%
head(11) %>%
mutate(Ratio = case_when(ONTOLOGY == "BP"~ Count/101,
ONTOLOGY == "MF"~ Count/107)) %>%
mutate(regulation = "Methylation")
go_ora_not_meth_plot <- as_tibble(go_ora_not_meth) %>%
arrange(desc(Count)) %>%
head(11) %>%
mutate(Ratio = case_when(ONTOLOGY == "BP"~ Count/76,
ONTOLOGY == "CC"~ Count/93))%>%
mutate(regulation = "Not methylation")
rbind(go_ora_meth_plot, go_ora_not_meth_plot) %>%
ggplot(aes(x = Ratio, y = Description, fill = Description)) +
geom_col() +
theme_bw() +
ylab("GO term") +
xlab("Gene Ratio") +
facet_wrap(~ regulation) +
theme(legend.position = "none",
axis.ticks.y = element_blank(),
axis.text.y = element_text(size = 6, color = "gray10"),
axis.title.x = element_text(size = 10, color = "gray10"),
axis.title.y = element_blank())+
geom_text(aes(0, y = Description, label = ID),
hjust = 0,
nudge_x = 0.005,
colour = "floralwhite",
size = 2)
We’ve also added a column tumor suppressor and oncogene in CTexploreR. These information come from Cancermine.
table(CT_genes$oncogene) ##
## oncogene
## 34table(CT_genes$tumor_suppressor)##
## tumor_suppressor
## 15table(CT_genes$oncogene, CT_genes$tumor_suppressor) ##
## tumor_suppressor
## oncogene 94 SessionInfo
sessionInfo()## R version 4.3.0 (2023-04-21)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 22.04.2 LTS
##
## Matrix products: default
## BLAS: /usr/lib/x86_64-linux-gnu/blas/libblas.so.3.10.0
## LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.10.0
##
## locale:
## [1] LC_CTYPE=C.UTF-8 LC_NUMERIC=C LC_TIME=C.UTF-8
## [4] LC_COLLATE=C.UTF-8 LC_MONETARY=C.UTF-8 LC_MESSAGES=C.UTF-8
## [7] LC_PAPER=C.UTF-8 LC_NAME=C LC_ADDRESS=C
## [10] LC_TELEPHONE=C LC_MEASUREMENT=C.UTF-8 LC_IDENTIFICATION=C
##
## time zone: Europe/Brussels
## tzcode source: system (glibc)
##
## attached base packages:
## [1] stats4 grid stats graphics grDevices utils datasets
## [8] methods base
##
## other attached packages:
## [1] Biostrings_2.70.1 XVector_0.42.0
## [3] patchwork_1.1.3 BiocParallel_1.36.0
## [5] DOSE_3.28.0 msigdbr_7.5.1
## [7] clusterProfiler_4.10.0 org.Hs.eg.db_3.18.0
## [9] AnnotationDbi_1.64.1 SingleCellExperiment_1.24.0
## [11] circlize_0.4.15 ComplexHeatmap_2.18.0
## [13] UpSetR_1.4.0 SummarizedExperiment_1.32.0
## [15] Biobase_2.62.0 GenomicRanges_1.54.1
## [17] GenomeInfoDb_1.38.1 IRanges_2.36.0
## [19] S4Vectors_0.40.1 MatrixGenerics_1.14.0
## [21] matrixStats_1.1.0 lubridate_1.9.3
## [23] forcats_1.0.0 stringr_1.5.1
## [25] dplyr_1.1.3 purrr_1.0.2
## [27] tidyr_1.3.0 tibble_3.2.1
## [29] ggplot2_3.4.4 tidyverse_2.0.0
## [31] biomaRt_2.58.0 Vennerable_3.0
## [33] xtable_1.8-4 gtools_3.9.4
## [35] reshape_0.8.9 RColorBrewer_1.1-3
## [37] lattice_0.22-5 RBGL_1.78.0
## [39] graph_1.80.0 BiocGenerics_0.48.1
## [41] CTexploreR_0.99.5 CTdata_1.2.0
## [43] readr_2.1.4 readxl_1.4.3
##
## loaded via a namespace (and not attached):
## [1] splines_4.3.0 later_1.3.1
## [3] ggplotify_0.1.2 bitops_1.0-7
## [5] filelock_1.0.2 cellranger_1.1.0
## [7] polyclip_1.10-6 XML_3.99-0.14
## [9] lifecycle_1.0.4 doParallel_1.0.17
## [11] vroom_1.6.4 MASS_7.3-60
## [13] magrittr_2.0.3 sass_0.4.7
## [15] rmarkdown_2.25 jquerylib_0.1.4
## [17] yaml_2.3.7 httpuv_1.6.12
## [19] cowplot_1.1.1 DBI_1.1.3
## [21] abind_1.4-5 zlibbioc_1.48.0
## [23] ggraph_2.1.0 RCurl_1.98-1.13
## [25] yulab.utils_0.1.0 tweenr_2.0.2
## [27] rappdirs_0.3.3 GenomeInfoDbData_1.2.11
## [29] enrichplot_1.22.0 ggrepel_0.9.4
## [31] tidytree_0.4.5 codetools_0.2-19
## [33] DelayedArray_0.28.0 xml2_1.3.5
## [35] ggforce_0.4.1 tidyselect_1.2.0
## [37] shape_1.4.6 aplot_0.2.2
## [39] farver_2.1.1 viridis_0.6.4
## [41] BiocFileCache_2.10.1 jsonlite_1.8.7
## [43] GetoptLong_1.0.5 ellipsis_0.3.2
## [45] tidygraph_1.2.3 iterators_1.0.14
## [47] foreach_1.5.2 tools_4.3.0
## [49] progress_1.2.2 treeio_1.26.0
## [51] HPO.db_0.99.2 Rcpp_1.0.11
## [53] glue_1.6.2 gridExtra_2.3
## [55] SparseArray_1.2.2 xfun_0.41
## [57] qvalue_2.34.0 withr_2.5.2
## [59] BiocManager_1.30.22 fastmap_1.1.1
## [61] fansi_1.0.5 digest_0.6.33
## [63] gridGraphics_0.5-1 timechange_0.2.0
## [65] R6_2.5.1 mime_0.12
## [67] colorspace_2.1-0 Cairo_1.6-1
## [69] GO.db_3.18.0 RSQLite_2.3.3
## [71] utf8_1.2.4 generics_0.1.3
## [73] data.table_1.14.8 prettyunits_1.2.0
## [75] graphlayouts_1.0.1 httr_1.4.7
## [77] S4Arrays_1.2.0 scatterpie_0.2.1
## [79] pkgconfig_2.0.3 gtable_0.3.4
## [81] blob_1.2.4 shadowtext_0.1.2
## [83] htmltools_0.5.7 fgsea_1.28.0
## [85] clue_0.3-65 scales_1.2.1
## [87] png_0.1-8 ggfun_0.1.3
## [89] knitr_1.45 rstudioapi_0.15.0
## [91] tzdb_0.4.0 reshape2_1.4.4
## [93] rjson_0.2.21 nlme_3.1-163
## [95] curl_5.1.0 cachem_1.0.8
## [97] GlobalOptions_0.1.2 BiocVersion_3.18.0
## [99] parallel_4.3.0 HDO.db_0.99.1
## [101] pillar_1.9.0 vctrs_0.6.4
## [103] promises_1.2.1 dbplyr_2.4.0
## [105] cluster_2.1.4 evaluate_0.23
## [107] magick_2.8.1 cli_3.6.1
## [109] compiler_4.3.0 rlang_1.1.2
## [111] crayon_1.5.2 labeling_0.4.3
## [113] fs_1.6.3 plyr_1.8.9
## [115] stringi_1.8.1 viridisLite_0.4.2
## [117] babelgene_22.9 MPO.db_0.99.7
## [119] munsell_0.5.0 lazyeval_0.2.2
## [121] GOSemSim_2.28.0 Matrix_1.6-1.1
## [123] ExperimentHub_2.10.0 hms_1.1.3
## [125] bit64_4.0.5 KEGGREST_1.42.0
## [127] shiny_1.7.5.1 highr_0.10
## [129] interactiveDisplayBase_1.40.0 AnnotationHub_3.10.0
## [131] igraph_1.5.1 memoise_2.0.1
## [133] bslib_0.5.1 ggtree_3.10.0
## [135] fastmatch_1.1-4 bit_4.0.5
## [137] gson_0.1.0 ape_5.7-1